EP0371262A1 - Digoxigenin derivatives and their use - Google Patents
Digoxigenin derivatives and their use Download PDFInfo
- Publication number
- EP0371262A1 EP0371262A1 EP89119938A EP89119938A EP0371262A1 EP 0371262 A1 EP0371262 A1 EP 0371262A1 EP 89119938 A EP89119938 A EP 89119938A EP 89119938 A EP89119938 A EP 89119938A EP 0371262 A1 EP0371262 A1 EP 0371262A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- digoxigenin
- formula
- group
- derivatives
- conjugates
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07J—STEROIDS
- C07J19/00—Normal steroids containing carbon, hydrogen, halogen or oxygen, substituted in position 17 by a lactone ring
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07J—STEROIDS
- C07J41/00—Normal steroids containing one or more nitrogen atoms not belonging to a hetero ring
- C07J41/0033—Normal steroids containing one or more nitrogen atoms not belonging to a hetero ring not covered by C07J41/0005
- C07J41/0055—Normal steroids containing one or more nitrogen atoms not belonging to a hetero ring not covered by C07J41/0005 the 17-beta position being substituted by an uninterrupted chain of at least three carbon atoms which may or may not be branched, e.g. cholane or cholestane derivatives, optionally cyclised, e.g. 17-beta-phenyl or 17-beta-furyl derivatives
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07J—STEROIDS
- C07J43/00—Normal steroids having a nitrogen-containing hetero ring spiro-condensed or not condensed with the cyclopenta(a)hydrophenanthrene skeleton
- C07J43/003—Normal steroids having a nitrogen-containing hetero ring spiro-condensed or not condensed with the cyclopenta(a)hydrophenanthrene skeleton not condensed
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6813—Hybridisation assays
- C12Q1/6816—Hybridisation assays characterised by the detection means
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
Definitions
- the invention relates to new digoxigenin derivatives and their use.
- Digoxigenin derivatives in which the basic structure of digoxigenin is covalently bound to another molecule or a macromolecular carrier, are used for a variety of bioanalytical purposes.
- digoxigenin derivatives are used in immunological tests (immunoassays) for determining cardiotonic glycosides, in particular digoxin, which are important with regard to therapeutic aspects (cf. GC Oliver et al, J.Clin.Invest. 47 (1968), 1035; U .Barbieri and C. Gandolfi, Clin. Chim. Acta 77 (1977), 257; A. Castro and N.
- the digoxigenin derivatives used are generally those derivatives in which the digoxigenin is covalently bound to the other molecule via the 3-position of the steroid structure.
- the digoxigenin derivatives When linked to the digoxigenin steroid structure via an ester group, the digoxigenin derivatives are very base-labile under basic conditions due to the sensitivity of the ester group to hydrolysis; this is particularly true for the commonly used hemisuccinates or hemiglutarates (cf. CH-PS 604 164; US-PS 4 082 747; N. Monji et al, Experientia 36 (1980) 1141); the same applies e.g. also for the urethane grouping used instead of the ester grouping (cf. DE-OS 26 07 826). Under certain conditions, the digoxigenin may be undesirably split off during use.
- digoxigenin In addition to the 3-position of the steroid structure, digoxigenin also has a reactive OH function in the 12-position; in the preparation of the derivatives, therefore, a mixture of 3 and 12 derivatives is often obtained, some of which can only be separated into the pure positional isomers with considerable preparative effort and some, e.g. in the case of the preferred hemisuccinates and hemiglutarates, it cannot be separated by chromatography. However, the use of the unseparated mixtures can lead to incorrect results.
- the steroid backbone is partially modified in such a way that recognition of the hapten can be severely impaired by an antibody directed against the unmodified steroid backbone.
- This can be the case, for example, with digoxigenin derivatives in which the oxygen atom in the 3-position is replaced by amino nitrogen (cf., for example, EP-B 104 527).
- the object of the present invention was to provide digoxigenin derivatives which are well suited for immunological tests for the determination of cardiotonic glycosides and with which the disadvantages described above can be avoided. This object is achieved with the present invention.
- the invention relates to digoxigenin derivatives of the general formula (I) wherein n represents an integer from 1 to 4, and preferably 1, and Z is a carboxy group or a functional derivative derived therefrom.
- Z is the group -COOH, -CN, -COOR5, -CONR1R2, -COONR3R4, -CON3, -COOCOO-R5 or -COOCOR5, wherein R1, R2, R3 and R4 independently of one another are a hydrogen atom, an alkyl group, an aralkyl group or are an aryl group, or R1 and R2 or R3 and R4 together can also form a carbocyclic or heterocyclic ring system.
- R5 is an alkyl or aralkyl group, preferably with 1 to 7 carbon atoms, in particular methyl, ethyl, propyl, isobutyl, phenyl, benzyl.
- an alkyl group can be branched or preferably straight-chain; the alkyl group preferably has 1 to 12 and in particular 2 to 8 carbon atoms.
- An alkyl group can be replaced by one or more heteroatoms such as e.g. Oxygen, interrupted and substituted or unsubstituted.
- An aryl group can be a mono- or polynuclear carbo- or heterocyclic aromatic radical with preferably 5 to 14 ring atoms; it can be substituted or unsubstituted and is especially phenyl.
- An aralkyl group is derived from the alkyl and aryl radicals defined above; it is e.g. a benzyl or phenylethyl group.
- the group -CONR1R2 is, for example, the radical -CONH ( ⁇ CH2CH2O) ⁇ 2CH2CH2NH2, in which the NH2 group can be substituted, preferably by 4-azidobenzoyl.
- the group -COONR3R4 is preferably the N-hydroxysuccinimide ester group
- An alkyl group R5 is in particular ethyl, isobutyl or benzyl.
- the bond between the steroid skeleton and the bridge takes place via an ether bond in the 3-position, as a result of which the bond with an ester group Base instability is avoided.
- This also maintains the 3-position function (oxy group) in the digoxigenin steroid framework, which ensures better detection of hapten by the antibody, which is usually directed against the unmodified steroid framework.
- the ether bond according to the invention in the 3-position also enables the compounds of the formula (I) and their derivatives and conjugates to be represented as pure position isomers, ie while avoiding a mixture of derivatives which are bonded in the 3- and 12-position.
- the compounds of the formula (I) according to the invention can be prepared starting from 12-O-acetyl-digoxigenin (compound 3 in the reaction scheme below), that from digoxin (compound 1) after conversion into the pentaacetyl digoxin (compound 2) and acid Saponification is available in a manner known per se.
- 12-O-acetyl-digoxigenin the free OH group in the 3-position is now converted into the ether group Z ( ⁇ CH2) ⁇ n O-.
- Z CONR1R2 (corresponds to compound 7) by reaction with an amine HNR1R2.
- Digoxigenin or digoxin is preferably used as the steroid. If the hapten is incorporated into the nucleic acid probe photochemically using a photo hapten (cf. Nucl.Acid Res. 13 (1985) 745 to 761; and M.Wilchek and EA Bayer, Anal. Bio chem. 171 (1988) 1), the preferred photo-hapten is photo-digoxigenin, ie a digoxigenin connected to the 4-azido-benzoyl residue via a bridge. When exposed to UV radiation, nitrogen is removed from the azido group to form a nitrene radical, which then covalently binds to the nucleic acid. The digoxigenin-3-hemisuccinate- [N ′ - (4-azidobenzoyl)] - 8-amino-3,6-dioxaoctylamide is used as the photo-digoxigenin.
- the digoxigenin derivatives of the formula (I) according to the invention are very suitable as labeling hapten for the above-mentioned method for the detection of nucleic acids.
- photo-hapten photo-digoxigenin; binding of the hapten by photochemical means, see Nucl. Acid Res. 13 (1985) 745 to 761; and M.Wilchek and EABayer, Anal. Biochem.
- digoxigenin derivatives according to the invention of the general formula (I) it is possible to link the 3-position of the steroid with the oxygen present there, that is to say without modifying the basic structure, via a bridge molecule to an immunogenic carrier material, such as a protein or polypeptide carrier material , or to bind to a nucleic acid.
- an immunogenic carrier material such as a protein or polypeptide carrier material
- the digoxigenin derivatives of the formula (I) according to the invention are also very suitable for the preparation of labeled conjugates, such as those used for Determination of cardiotonic glycosides, in particular digoxin, can be used in conventional immunoassays.
- Suitable conjugates can, for example, be radioactively labeled in accordance with standard methods or labeled with a fluorescent grouping.
- the labeled structural unit (labeling moiety) is, for example, an enzyme substrate, a prosthetic group, an enzyme modulator or an enzyme which is coupled to the conjugate with the digoxigenin derivatives of the formula (I) according to the invention.
- Binding to the support material, to the nucleic acid or to the marked structural unit takes place via the grouping -0- (CH2) n -Z, the grouping Z preferably being selected depending on the support material and the groups or bonds reacting with Z.
- the grouping -0- (CH2) n -Z can also by the photochemical method (cf. Nucl.Acid Res. 13 (1985) 745 to 761; and M.Wilchek and EA Bayer, Anal.Biochem. 171 (1988) 1) bound to the support, for example the nucleic acid;
- the carrier material for example the nucleic acid probe
- the carrier material is irradiated in the presence of the photo-digoxigenin mentioned above (see reaction scheme, compound 7) with visible light with a UV component, a nitrenic radical being formed with the elimination of nitrogen (N2) binds covalently to the nucleic acid.
- the invention therefore also relates to the use of digoxigenin derivatives of the general formula (I) for the preparation of labeled conjugates for the determination of cardiotonic glycosides, in particular digoxin, in conventional immunoassays and their use as marker hapten for the detection of nucleic acids Hybridization with a complementary nucleic acid probe which contains at least one hapten bound as a label via a chemical compound.
- the invention also relates to the new digoxigenin conjugates of the formula (II) prepared using the digoxigenin derivatives of the formula (I) according to the invention.
- the carrier is an immunogenic carrier material such as an immunogenic protein or polypeptide carrier material, a nucleic acid or the labeled structural unit of a labeled digoxigenin conjugate for use in immunoassays, y on average a number from 1 to the number of those in the carrier available coupling sites, and is preferably 1 to 20, and Y is the reaction of the carboxyl function Z of the digoxigenin derivatives of the formula (I) according to the invention with the reacting sites of the support material is a group, for example an amide group.
- Another object of the invention is the use of the digoxigenin conjugates of the formula (II), in which the carrier is an immunogen, for the immunization of organisms suitable for antibody formation.
- the digoxigenin conjugates of the formula (II), in which the carrier is an immunogen can be used to produce antibodies, which can then be used in one of the customary immunoassay methods for the determination of digoxin (eg agglutination techniques, radioimmunoassays, heterogeneous enzyme immunoassays , heterogeneous fluorescence immunoassays, and homogeneous immunoassays) can be used.
- These antibodies, including monoclonal antibodies can be prepared and isolated in a manner which is customary and generally known.
- Another object is the use of the digoxigenin conjugates of the formula (II), in which the carrier is the labeled structural unit of a labeled digoxigenin conjugate, in conventional immunoassays for the determination of cardiotonic glycosides, in particular digoxin.
- reaction scheme A gives an overview of the synthesis of the digoxigenin derivatives of the formula (I) according to the invention shown in the examples and their mutual conversion.
- Example 2 The evaporation residue obtained according to Example 1 (compound 2) is dissolved in 2 l of methanol, 2 l of 0.1 N H2SO4 and stirred for 1 hour under reflux. Then it is extracted once with 1.8 l and once with 600 ml of chloroform, the combined extracts are washed twice with 1 l of water, dried with 50 g of Na2SO4 and evaporated in a water jet vacuum. The oil obtained (95 g) is dissolved in 250 ml of ethyl acetate with gentle heating and left to stand at room temperature. Crystallization occurs after a short time.
- reaction scheme B shows schematically the step-by-step production of Dig-11-dUTP, which can be used, for example, as marker hapten for the detection of nucleic acids by hybridization with a complementary, labeled nucleic acid, according to Examples 7 to 9.
- the reaction is according to thin layer chromatography (silica gel; ethyl acetate / petroleum ether / ethanol 1: 1: 1, detection: spraying with a mixture of 10 ml glacial acetic acid + 0.2 ml conc. H2SO4 + 0.1 ml anisaldehyde and heating to 120 ° C until blue-black spots appear; R f approx. 0.7; R f digoxigenin-OSu-ester approx. 0.85) practically completely.
- reaction mixture is evaporated in an oil pump vacuum to a solid residue, taken up in 200 ml of H2O and on an ion exchange column (DEAE-Sephadex A25, HCO 3 Shape, column dimension 1.5 x 30 cm).
- ion exchange column DEAE-Sephadex A25, HCO 3 Shape, column dimension 1.5 x 30 cm.
- the mixture is washed briefly with water, then eluted with a gradient of 1 l H2O to 0.4 mol / l TEAB (triethylammonium bicarbonate), pH 8.
- the fractions containing pure product are combined, concentrated in vacuo and freed of excess TEAB by repeated evaporation with methanol (no more odor of free triethylamine!).
- Dig-11-dUTP can be carried out according to the "random primed" DNA labeling method (DNA labeling and detection kit non-radioactive, order no. 1093657, from Boehringer Mannheim; Feinberg, AP and Vogelstein, B., Anal. Biochem. 132 , (1983) 6) are introduced into a nucleic acid, a nucleic acid probe containing digoxigenin as labeling hapten being obtained for the detection of complementary nucleic acids.
- DNA labeling and detection kit non-radioactive, order no. 1093657 from Boehringer Mannheim; Feinberg, AP and Vogelstein, B., Anal. Biochem. 132 , (1983) 6
- DNA labeling and detection kit non-radioactive, order no. 1093657 from Boehringer Mannheim; Feinberg, AP and Vogelstein, B., Anal. Biochem. 132 , (1983) 6
- DNA labeling and detection kit non-radioactive, order no. 1093657 from Boehringer Mannheim
Abstract
Description
Die Erfindung betrifft neue Digoxigenin-Derivate und ihre Verwendung.The invention relates to new digoxigenin derivatives and their use.
Digoxigenin-Derivate, bei denen das Grundgerüst des Digoxigenins kovalent an ein anderes Molekül oder einen makromolekularen Träger gebunden vorliegt, werden für eine Vielzahl von bioanalytischen Zwecken verwendet. Insbesondere werden solche Digoxigenin-Derivate in immunologischen Tests (Immunoassays) zur im Hinblick auf therapeutische Aspekte wichtigen Bestimmung von kardiotonischen Glycosiden, insbesondere Digoxin, eingesetzt (vgl. G.C. Oliver et al, J.Clin.Invest. 47 (1968), 1035; U.Barbieri und C. Gandolfi, Clin.Chim. Acta 77 (1977), 257; A. Castro und N. Monji, "Immunochemical Methods", Teil B, herausgegeben von J.L. Langone und H.van Vunakis, Academic Press 1981, S. 523; J.A. Hinds et al, Clinical Chemistry 32 (1986) 16). In diesen Tests werden die Digoxigenin-Derivate in Verbindung mit Antikörpern gegen die zu bestimmenden kardiotonischen Glycoside eingesetzt, und der Nachweis erfolgt auf der Grundlage des Prinzips der Hapten-Anti-HaptenAntikörper-Wechselwirkung (vgl. K. Luebke und B. Nieuweboer, "Immunologische Teste für niedermolekulare Wirkstoffe", Thieme Verlag Stuttgart, 1978).Digoxigenin derivatives, in which the basic structure of digoxigenin is covalently bound to another molecule or a macromolecular carrier, are used for a variety of bioanalytical purposes. In particular, such digoxigenin derivatives are used in immunological tests (immunoassays) for determining cardiotonic glycosides, in particular digoxin, which are important with regard to therapeutic aspects (cf. GC Oliver et al, J.Clin.Invest. 47 (1968), 1035; U .Barbieri and C. Gandolfi, Clin. Chim. Acta 77 (1977), 257; A. Castro and N. Monji, "Immunochemical Methods", Part B, edited by JL Langone and H.van Vunakis, Academic Press 1981, p 523; JA Hinds et al, Clinical Chemistry 32 (1986) 16). In these tests, the digoxigenin derivatives are used in conjunction with antibodies against the cardiotonic glycosides to be determined, and the detection is based on the principle of the hapten-anti-hapten-antibody interaction (cf. K. Luebke and B. Nieuweboer, "Immunological Tests for low molecular weight active substances ", Thieme Verlag Stuttgart, 1978).
Als Digoxigenin-Derivate werden dabei im allgemeinen solche Derivate verwendet, in denen das Digoxigenin über die 3-Stellung des Steroidgerüstes kovalent an das andere Molekül gebunden ist.The digoxigenin derivatives used are generally those derivatives in which the digoxigenin is covalently bound to the other molecule via the 3-position of the steroid structure.
Die in den immunologischen Tests bisher verwendeten Digoxigenin-Derivate weisen jedoch einen oder mehrere der folgenden Nachteile auf:However, the digoxigenin derivatives previously used in the immunological tests have one or more of the following disadvantages:
Bei einer über eine Estergruppierung erfolgenden Verknüpfung mit dem Digoxigenin-Steroidgerüst sind die Digoxigenin-Derivate aufgrund der Hydrolyseempfindlichkeit der Estergruppierung unter basischen Bedingungen sehr basenlabil; dies trifft im besonderen Maße für die üblicherweise verwendeten Hemisuccinate oder Hemiglutarate (vgl. CH-PS 604 164; US-PS 4 082 747; N. Monji et al, Experientia 36 (1980) 1141) zu; ähnliches gilt z.B. auch für die anstelle der Estergruppierung verwendete Urethangruppierung (vgl. DE-OS 26 07 826). Bei der Anwendung kann es deshalb unter bestimmten Bedingungen zu einer unerwünschten Abspaltung des Digoxigenins kommen.When linked to the digoxigenin steroid structure via an ester group, the digoxigenin derivatives are very base-labile under basic conditions due to the sensitivity of the ester group to hydrolysis; this is particularly true for the commonly used hemisuccinates or hemiglutarates (cf. CH-PS 604 164; US-PS 4 082 747; N. Monji et al, Experientia 36 (1980) 1141); the same applies e.g. also for the urethane grouping used instead of the ester grouping (cf. DE-OS 26 07 826). Under certain conditions, the digoxigenin may be undesirably split off during use.
Außer in der 3-Stellung des Steroidgerüstes besitzt Digoxigenin auch in der 12-Stellung eine reaktive OH-Funktion; bei der Herstellung der Derivate fällt deshalb oft ein Gemisch aus 3- und 12-Derivat an, das sich zum Teil nur unter erheblichem präparativen Aufwand in die reinen Stellungsisomeren auftrennen läßt und zum Teil, z.B. im Fall der bevorzugt verwendeten Hemisuccinate und Hemiglutarate, chromatographisch nicht auftrennbar ist. Die Anwendung der nicht aufgetrennten Gemische kann aber zu fehlerhaften Ergebnissen führen.In addition to the 3-position of the steroid structure, digoxigenin also has a reactive OH function in the 12-position; in the preparation of the derivatives, therefore, a mixture of 3 and 12 derivatives is often obtained, some of which can only be separated into the pure positional isomers with considerable preparative effort and some, e.g. in the case of the preferred hemisuccinates and hemiglutarates, it cannot be separated by chromatography. However, the use of the unseparated mixtures can lead to incorrect results.
In den Derivaten liegt das Steroidgrundgerüst zum Teil derart modifiziert vor, daß eine Erkennung des Haptens durch einen gegen das unmodifizierte Steroidgrundgerüst gerichteten Antikörper stark beeinträchtigt sein kann. Dies kann z.B. bei Digoxigenin-Derivaten, in denen das Sauerstoffatom in der 3-Stellung durch Amino-Stickstoff ersetzt ist, der Fall sein (vgl. z.B. EP-B 104 527).In the derivatives, the steroid backbone is partially modified in such a way that recognition of the hapten can be severely impaired by an antibody directed against the unmodified steroid backbone. This can be the case, for example, with digoxigenin derivatives in which the oxygen atom in the 3-position is replaced by amino nitrogen (cf., for example, EP-B 104 527).
Aufgabe der vorliegenden Erfindung war es, Digoxigenin-Derivate bereitzustellen, die sich gut für immunologische Tests zur Bestimmung von kardiotonischen Glycosiden eignen und mit denen sich die vorstehend beschriebenen Nachteile vermeiden lassen. Diese Aufgabe wird mit der vorliegenden Erfindung gelöst.The object of the present invention was to provide digoxigenin derivatives which are well suited for immunological tests for the determination of cardiotonic glycosides and with which the disadvantages described above can be avoided. This object is achieved with the present invention.
Gegenstand der Erfindung sind Digoxigenin-Derivate der allgemeinen Formel (I)
R⁵ ist eine Alkyl- oder Aralkylgruppe, vorzugsweise mit 1 bis 7 Kohlenstoffatomen, insbesondere Methyl, Ethyl, Propyl, Isobutyl, Phenyl, Benzyl.R⁵ is an alkyl or aralkyl group, preferably with 1 to 7 carbon atoms, in particular methyl, ethyl, propyl, isobutyl, phenyl, benzyl.
In den vorstehend genannten Resten kann eine Alkylgruppe verzweigt oder vorzugsweise geradkettig sein; vorzugsweise besitzt die Alkylgruppe 1 bis 12, und insbesondere 2 bis 8 Kohlenstoffatome. Eine Alkylgruppe kann durch ein oder mehrere Heteroatome wie z.B. Sauerstoff, unterbrochen sein und substituiert oder unsubstituiert sein.In the radicals mentioned above, an alkyl group can be branched or preferably straight-chain; the alkyl group preferably has 1 to 12 and in particular 2 to 8 carbon atoms. An alkyl group can be replaced by one or more heteroatoms such as e.g. Oxygen, interrupted and substituted or unsubstituted.
Eine Arylgruppe kann ein ein- oder mehrkerniger carbo- oder heterocyclischer aromatischer Rest mit vorzugsweise 5 bis 14 Ringatomen sein; sie kann substituiert oder unsubstituiert sein und ist insbesondere Phenyl. Eine Aralkylgruppe leitet sich von den vorstehend definierten Alkyl- und Arylresten ab; sie ist z.B. eine Benzyl- oder Phenylethyl-Gruppe.An aryl group can be a mono- or polynuclear carbo- or heterocyclic aromatic radical with preferably 5 to 14 ring atoms; it can be substituted or unsubstituted and is especially phenyl. An aralkyl group is derived from the alkyl and aryl radicals defined above; it is e.g. a benzyl or phenylethyl group.
Die Gruppierung -CONR¹R² ist z.B. der Rest -CONH(̵CH₂CH₂O)̵₂CH₂CH₂NH₂, in dem die NH₂-Gruppe substituiert sein kann, vorzugsweise durch 4-Azidobenzoyl. Die Gruppe -COONR³R⁴ ist vorzugsweise die N-Hydroxysuccinimidestergruppierung
Eine Alkylgruppe R⁵ ist insbesondere Ethyl, Isobutyl oder Benzyl.An alkyl group R⁵ is in particular ethyl, isobutyl or benzyl.
In den erfindungsgemäßen Digoxigenin-Derivaten der Formel (I) erfolgt die Bindung zwischen dem Steroidgerüst und der Brücke über eine Etherbindung in 3-Stellung, wodurch die mit einer Estergruppierung verbundene Basenlabilität vermieden wird. Außerdem wird dadurch die im Digoxigenin-Steroidgerüst in 3-Stellung vorhandene Funktion (Oxy-Gruppe) aufrechterhalten, wodurch eine bessere Erkennung des Haptens durch den Antikörper, der in der Regel gegen das nicht modifizierte Steroidgerüst gerichtet ist, gewährleistet wird. Die erfindungsgemäße Etherbindung in 3-Stellung ermöglicht auch die Darstellung der Verbindungen der Formel (I) und deren Derivate und Konjugate als reine Stellungsisomere, d.h. unter Vermeidung eines Gemisches aus Derivaten, die in 3- und 12-Stellung gebunden sind.In the digoxigenin derivatives of the formula (I) according to the invention, the bond between the steroid skeleton and the bridge takes place via an ether bond in the 3-position, as a result of which the bond with an ester group Base instability is avoided. This also maintains the 3-position function (oxy group) in the digoxigenin steroid framework, which ensures better detection of hapten by the antibody, which is usually directed against the unmodified steroid framework. The ether bond according to the invention in the 3-position also enables the compounds of the formula (I) and their derivatives and conjugates to be represented as pure position isomers, ie while avoiding a mixture of derivatives which are bonded in the 3- and 12-position.
Die Herstellung der erfindungsgemäßen Verbindungen der Formel (I) kann ausgehend von 12-O-Acetyl-digoxigenin (im nachstehenden Reaktionsschema die Verbindung 3) erfolgen, daß aus Digoxin (Verbindung 1) nach Überführung in das Pentaacetyl-digoxin (Verbindung 2) und saure Verseifung auf an sich bekannte Weise erhältlich ist. Im 12-O-Acetyl-digoxigenin wird nun die freie OH-Gruppe in 3-Stellung in die Ethergruppierung Z(̵CH₂)̵nO- überführt. Diese Umsetzung kann mit einem Diazocarbonsäureester, für n = 1 z.B. mit Diazoessigester, auf an sich bekannte Weise unter Bildung des 3-Alkoxycarbonylalkylethers erfolgen (Z = COOR⁵; entspricht Verbindung 4). In dem erhaltenen Alkoxycarbonylalkylester kann, wenn erwünscht, die Estergruppierung durch Verseifung in die freie Carboxygruppe (Z = COOH; Verbindung 5) oder eine andere funktionelle Carbonsäuregruppierung Z überführt werden, und, wenn erwünscht, kann in dem so erhaltenen Reaktionsprodukt die Gruppe Z auf an sich bekannte Weise in eine andere Gruppe Z überführt werden. Durch Umsetzung mit N-Hydroxysuccinimid kann die COOH-Gruppe z.B. in die Gruppierung -COONR³R⁴ (R³ und R⁴ bilden zusammen den 1,4-Dioxo-tetramethylen-Rest; Verbindung 6) überführt werden; diese Gruppierung läßt sich durch Umsetzung mit einem Amin HNR¹R² in die Verbindung (I) mit Z = CONR¹R² (entspricht Verbindung 7) überführen. Auf diese oder ähnliche, an sich bekannte Weise ist es möglich, eine erhaltene Verbindung der Formel (I), in der Z eine der angegebenen Bedeutungen besitzt, in eine andere Verbindung der Formel (I) mit einer anderen Bedeutung von Z zu überführen.The compounds of the formula (I) according to the invention can be prepared starting from 12-O-acetyl-digoxigenin (compound 3 in the reaction scheme below), that from digoxin (compound 1) after conversion into the pentaacetyl digoxin (compound 2) and acid Saponification is available in a manner known per se. In 12-O-acetyl-digoxigenin, the free OH group in the 3-position is now converted into the ether group Z (̵CH₂) ̵ n O-. This reaction can be carried out with a diazocarboxylic acid ester, for n = 1, for example with diazoacetic ester, in a manner known per se to form the 3-alkoxycarbonylalkyl ether (Z = COOR⁵; corresponds to compound 4). In the resulting alkoxycarbonyl alkyl ester, if desired, the ester group can be converted to the free carboxy group (Z = COOH; compound 5) or another functional carboxylic acid group Z by saponification, and if desired the group Z can be added to the reaction product thus obtained can be converted into another group Z in a known manner. By reaction with N-hydroxysuccinimide, the COOH group can be converted, for example, into the grouping -COONR³R⁴ (R³ and R⁴ together form the 1,4-dioxotetramethylene radical; compound 6); this grouping leaves convert into compound (I) with Z = CONR¹R² (corresponds to compound 7) by reaction with an amine HNR¹R². In this or similar manner known per se, it is possible to convert a compound of the formula (I) in which Z has one of the meanings obtained into another compound of the formula (I) with a different meaning of Z.
Die einzelnen, vorstehend genannten Verfahrensschritte, wie z.B. die Umsetzung mit Diazoessigester, die Verseifungsstufen, die Umsetzung mit N-Hydroxysuccinimid und die Umsetzung mit einem Amin HNR¹R² kann auf eine für diese Umsetzung übliche Weise erfolgen, wie z.B. in den nachfolgenden Beispielen erläutert.The individual process steps mentioned above, e.g. the reaction with diazoacetic ester, the saponification stages, the reaction with N-hydroxysuccinimide and the reaction with an amine HNR¹R² can be carried out in a manner customary for this reaction, e.g. explained in the following examples.
Ein weiteres wichtiges Einsatzgebiet für Digoxigenin-Derivate ist auch ihre Verwendung zur Markierung und Detektion von Nucleinsäuren und Nucleinsäurederivaten. In den deutschen Patentanmeldungen der gleichen Anmelderin P 38 00 644.8 (vom 12. Januar 1988) und P 38 13 278.8 (vom 20. April 1988) wird ein Verfahren zum Nachweis von Nucleinsäuren durch Hybridisierung mit einer komplementären Nucleinsäure-Sonde, die über eine chemische Verbindung mindestens ein Hapten als Markierung gebunden enthält, beschrieben. Als Hapten verwendet man ein Steroid, das an mindestens eine Position der Nucleinsäure-Sonde, die nicht an der Wasserstoffbrückenbildung beteiligt ist, über eine Brücke von mindestens 4 Atomen Länge gebunden ist; die hybridisierte Sonde weist man über einen markierten Anti-Hapten-Antikörper nach. Als Steroid wird vorzugsweise Digoxigenin oder Digoxin verwendet. Wird das Hapten in die Nucleinsäure-Sonde fotochemisch mit Hilfe eines Foto-Haptens eingebaut (vgl. Nucl.Acid Res. 13 (1985) 745 bis 761; und M.Wilchek und E.A. Bayer, Anal. Bio chem. 171 (1988) 1), so verwendet man als Foto-Hapten vorzugsweise Foto-Digoxigenin, d.h. ein über eine Brücke mit dem 4-Azido-benzoylrest verbundenes Digoxigenin. Bei UV-Bestrahlung entsteht unter Abspaltung von Stickstoff aus der Azidogruppe ein Nitrenradikal, das dann kovalent an die Nucleinsäure bindet. Als Foto-Digoxigenin wird das Digoxigenin-3-hemisuccinat-[N′-(4-azidobenzoyl)]-8-amino-3,6-dioxaoctylamid verwendet.Another important area of application for digoxigenin derivatives is their use for the labeling and detection of nucleic acids and nucleic acid derivatives. In the German patent applications of the same applicant P 38 00 644.8 (from January 12, 1988) and P 38 13 278.8 (from April 20, 1988) a method for the detection of nucleic acids by hybridization with a complementary nucleic acid probe, which is via a chemical Compound contains at least one hapten bound as a label. A steroid is used as the hapten, which is bound to at least one position of the nucleic acid probe which is not involved in the hydrogen bonding via a bridge of at least 4 atoms in length; the hybridized probe is detected using a labeled anti-hapten antibody. Digoxigenin or digoxin is preferably used as the steroid. If the hapten is incorporated into the nucleic acid probe photochemically using a photo hapten (cf. Nucl.Acid Res. 13 (1985) 745 to 761; and M.Wilchek and EA Bayer, Anal. Bio chem. 171 (1988) 1), the preferred photo-hapten is photo-digoxigenin, ie a digoxigenin connected to the 4-azido-benzoyl residue via a bridge. When exposed to UV radiation, nitrogen is removed from the azido group to form a nitrene radical, which then covalently binds to the nucleic acid. The digoxigenin-3-hemisuccinate- [N ′ - (4-azidobenzoyl)] - 8-amino-3,6-dioxaoctylamide is used as the photo-digoxigenin.
Aufgrund ihrer Eigenschaften, insbesondere aufgrund der Basenstabilität und des unmodifizierten Steroidgerüstes, eignen sich die erfindungsgemäßen Digoxigenin-Derivate der Formel (I) sehr gut als Markierungshapten für das vorstehend genannte Verfahren zum Nachweis von Nucleinsäuren. Als Foto-Hapten (Foto-Digoxigenin; Bindung des Haptens auf fotochemischem Wege, vgl. Nucl. Acid Res. 13 (1985) 745 bis 761; und M.Wilchek und E.A.Bayer, Anal. Biochem. 171 (1988) 1) wird ein erfindungsgemäßes Digoxigenin-Derivat der Formel (I) verwendet, worin einer der Reste R¹, R², R³, R⁴ und/oder R⁵ eine 4-Azidobenzoyl-Gruppe trägt, wie z.B. das N-[N-(4-Azidobenzoyl)-8-amino-3,6-dioxaoctyl] -3-carbamoylmethyl-digoxigenin (Verbindung 7 des Reaktionsschemas).Due to their properties, in particular due to the base stability and the unmodified steroid structure, the digoxigenin derivatives of the formula (I) according to the invention are very suitable as labeling hapten for the above-mentioned method for the detection of nucleic acids. As photo-hapten (photo-digoxigenin; binding of the hapten by photochemical means, see Nucl. Acid Res. 13 (1985) 745 to 761; and M.Wilchek and EABayer, Anal. Biochem. 171 (1988) 1) a digoxigenin derivative according to the invention of formula (I) used, wherein one of the radicals R¹, R², R³, R⁴ and / or R⁵ carries a 4-azidobenzoyl group, such as the N- [N- (4-azidobenzoyl) -8-amino-3,6-dioxaoctyl] -3-carbamoylmethyl digoxigenin (compound 7 of the reaction scheme).
Mit den erfindungsgemäßen Digoxigenin-Derivaten der allgemeinen Formel (I) ist es möglich, die 3-Position des Steroids mit dem dort vorhandenen Sauerstoff, also ohne Modifizierung des Grundgerüstes, über ein Brückenmolekül an ein immunogenes Trägermaterial, wie ein Protein- oder Polypeptid-Trägermaterial, oder an eine Nucleinsäure zu binden.With the digoxigenin derivatives according to the invention of the general formula (I) it is possible to link the 3-position of the steroid with the oxygen present there, that is to say without modifying the basic structure, via a bridge molecule to an immunogenic carrier material, such as a protein or polypeptide carrier material , or to bind to a nucleic acid.
Die erfindungsgemäßen Digoxigenin-Derivate der Formel (I) eignen sich auch sehr gut zur Herstellung von markierten Konjugaten (labeled conjugates), wie sie zur Bestimmung von kardiotonischen Glycosiden, insbesondere von Digoxin, in üblichen Immunoassays verwendet werden. Geeignete Konjugate können z.B. in Übereinstimmung mit Standardmethoden radioaktiv markiert oder mit einer Fluoreszenz-Gruppierung markiert sein. Die markierte Struktureinheit (labeling moiety) ist in den bevorzugten homogenen Techniken z.B. ein Enzymsubstrat, eine prostetische Gruppe ein Enzymmodulator oder ein Enzym, das mit den erfindungsgemäßen Digoxigenin-Derivaten der Formel (I) zum Konjugat gekuppelt ist.The digoxigenin derivatives of the formula (I) according to the invention are also very suitable for the preparation of labeled conjugates, such as those used for Determination of cardiotonic glycosides, in particular digoxin, can be used in conventional immunoassays. Suitable conjugates can, for example, be radioactively labeled in accordance with standard methods or labeled with a fluorescent grouping. In the preferred homogeneous techniques, the labeled structural unit (labeling moiety) is, for example, an enzyme substrate, a prosthetic group, an enzyme modulator or an enzyme which is coupled to the conjugate with the digoxigenin derivatives of the formula (I) according to the invention.
Die Bindung an das Trägermaterial, an die Nucleinsäure oder an die markierte Struktureinheit erfolgt über die Gruppierung -0-(CH₂)n-Z, wobei die Gruppierung Z vorzugsweise abhängig vom Trägermaterial und den mit Z reagierenden Gruppen oder Bindungen ausgewählt wird. Vorzugsweise erfolgt die Verknüpfung des Trägermaterials mit der -0-(CH₂)n-Z-Gruppierung über primäre oder sekundäre Aminogruppen, die mit einer aktivierten Carbonsäurefunktion Z umgesetzt werden, z.B. mit dem N-Hydroxysuccinimid-ester (Z = -COONR³R⁴, R³ und R⁴ bilden zusammen den 1,4-Dioxotetramethylen-Rest); die Umsetzung erfolgt dabei auf für eine solche Reaktion an sich bekannte Weise. Die Gruppierung -0-(CH₂)n-Z kann aber auch nach der fotochemischen Methode (vgl. Nucl.Acid Res. 13 (1985) 745 bis 761; und M.Wilchek und E.A. Bayer, Anal.Biochem. 171 (1988) 1) an den Träger, z.B. die Nucleinsäure, gebunden werden; in diesem Fall wird das Trägermaterial, z.B. die Nucleinsäure-Sonde, in Gegenwart des vorstehend genannten Foto-Digoxigenins (siehe Reaktionsschema, Verbindung 7) mit sichtbarem Licht mit UV-Anteil bestrahlt, wobei unter Abspaltung von Stickstoff (N₂) ein Nitrenradikal entsteht, das kovalent an die Nucleinsäure bindet.Binding to the support material, to the nucleic acid or to the marked structural unit takes place via the grouping -0- (CH₂) n -Z, the grouping Z preferably being selected depending on the support material and the groups or bonds reacting with Z. Preferably, the linkage of the carrier material with the -0- (CH₂) n -Z grouping takes place via primary or secondary amino groups which are reacted with an activated carboxylic acid function Z, for example with the N-hydroxysuccinimide ester (Z = -COONR³R⁴, R³ and R⁴ together form the 1,4-dioxotetramethylene residue); the implementation takes place in a manner known per se for such a reaction. The grouping -0- (CH₂) n -Z can also by the photochemical method (cf. Nucl.Acid Res. 13 (1985) 745 to 761; and M.Wilchek and EA Bayer, Anal.Biochem. 171 (1988) 1) bound to the support, for example the nucleic acid; In this case, the carrier material, for example the nucleic acid probe, is irradiated in the presence of the photo-digoxigenin mentioned above (see reaction scheme, compound 7) with visible light with a UV component, a nitrenic radical being formed with the elimination of nitrogen (N₂) binds covalently to the nucleic acid.
Gegenstand der Erfindung ist deshalb auch die Verwendung von Digoxigenin-Derivaten der allgemeinen Formel (I) zur Herstellung von markierten Konjugaten (labeled conjugates) zur Bestimmung von kardiotonischen Glycosiden, insbesondere von Digoxin, in üblichen Immunoassays und ihre Verwendung als Markerhapten zum Nachweis von Nucleinsäuren durch Hybridisierung mit einer komplementären Nucleinsäure-Sonde, die über eine chemische Verbindung mindestens ein Hapten als Markierung gebunden enthält.The invention therefore also relates to the use of digoxigenin derivatives of the general formula (I) for the preparation of labeled conjugates for the determination of cardiotonic glycosides, in particular digoxin, in conventional immunoassays and their use as marker hapten for the detection of nucleic acids Hybridization with a complementary nucleic acid probe which contains at least one hapten bound as a label via a chemical compound.
Ein weiterer Gegenstand der Erfindung sind auch die unter Verwendung der erfindungsgemäßen Digoxigenin-Derivate der Formel (I) hergestellten neuen Digoxigenin-Konjugate der Formel (II)
Weiterer Gegenstand der Erfindung ist auch die Verwendung der Digoxigenin-Konjugate der Formel (II), worin der Träger ein Immunogen darstellt, zur Immunisierung von zur Antikörper-Bildung geeigneten Organismen. Auf diese Weise können mit den erfindungsgemäßen Digoxigenin-Konjugaten der Formel (II), worin der Träger ein Immunogen darstellt, Antikörper hergestellt werden, die dann in einem der üblichen Immunoassay-Verfahren zur Bestimmung von Digoxin (z.B. Agglutinationstechniken, Radioimmunoassays, heterogene Enzym-Immunoassays, heterogene Fluoreszenz-Immunoassays, und homogene Immunoassays) eingesetzt werden können. Die Herstellung und Isolierung dieser Antikörper, einschließlich monoklonaler Antikörper kann auf eine dafür übliche und allgemein bekannte Weise erfolgen.Another object of the invention is the use of the digoxigenin conjugates of the formula (II), in which the carrier is an immunogen, for the immunization of organisms suitable for antibody formation. In this way, the digoxigenin conjugates of the formula (II), in which the carrier is an immunogen, can be used to produce antibodies, which can then be used in one of the customary immunoassay methods for the determination of digoxin (eg agglutination techniques, radioimmunoassays, heterogeneous enzyme immunoassays , heterogeneous fluorescence immunoassays, and homogeneous immunoassays) can be used. These antibodies, including monoclonal antibodies, can be prepared and isolated in a manner which is customary and generally known.
Ein weiterer Gegenstand ist auch die Verwendung der Digoxigenin-Konjugate der Formel (II), in denen der Träger die markierte Struktureinheit eines markierten Digoxigenin-Konjugates darstellt, in üblichen Immunoassays zur Bestimmung von kardiotonischen Glycosiden, insbesondere von Digoxin.Another object is the use of the digoxigenin conjugates of the formula (II), in which the carrier is the labeled structural unit of a labeled digoxigenin conjugate, in conventional immunoassays for the determination of cardiotonic glycosides, in particular digoxin.
Die nachfolgenden Beispiele sollten die Erfindung näher erläutern, ohne sie darauf zu beschränken. Wenn nicht anders angegeben, beziehen sich die Mengenangaben auf Gewichtsteile und Temperaturangaben auf °C. Unter Raumtemperatur wird eine Temperatur von 25 ± 2°C verstanden.The following examples should explain the invention in more detail without restricting it. Unless otherwise stated, the quantities given are in parts by weight and the temperatures are in ° C. A room temperature is understood to be a temperature of 25 ± 2 ° C.
Das nachfolgende Reaktionsschema A gibt einen Überblick über die in den Beispielen gezeigte Synthese der erfindungsgemäßen Digoxigenin-Derivate der Formel (I) und deren gegenseitige Überführung.
78 g (0,1 mol) Digoxin werden in 1 1 Acetanhydrid gelöst und mit 49,2 g (0,6 mol) Natriumacetat (wasserfrei) versetzt. Man läßt 1 Stunde unter Rückfluß rühren. Dann dampft man die Lösung am Wasserstrahlvakuum ein, löst den Rückstand in 1 l Essigester und filtriert von eventuell unlöslichen Bestandteilen ab. Das Filtrat wird dreimal mit je 0,5 l Wasser gewaschen, mit 50 g Na₂SO₄ getrocknet und im Wasserstrahlvakuum eingedampft. Das Rohprodukt enthält noch Acetanhydrid.
Ausbeute: 110 g zähes Öl.
DC: Kieselgel, Essigester/Chloroform 1:1; Rf = 0,33.78 g (0.1 mol) of digoxin are dissolved in 1 l of acetic anhydride and 49.2 g (0.6 mol) of sodium acetate (anhydrous) are added. The mixture is stirred under reflux for 1 hour. Then the solution is evaporated in a water jet vacuum, the residue is dissolved in 1 l of ethyl acetate and any insoluble constituents are filtered off. The filtrate is washed three times with 0.5 l of water, dried with 50 g Na₂SO₄ and evaporated in a water jet vacuum. The crude product still contains acetic anhydride.
Yield: 110 g of viscous oil.
TLC: silica gel, ethyl acetate / chloroform 1: 1; R f = 0.33.
Der nach Beispiel 1 erhaltene Eindampfrückstand (Verbindung 2) wird in 2 l Methanol gelöst, mit 2 l 0,1 n H₂SO₄ versetzt und l Stunde unter Rückfluß geruhrt. Danach wird einmal mit 1,8 l und einmal mit 600 ml Chloroform extrahiert, die vereinigten Extrakte zweimal mit je 1 l Wasser gewaschen, mit 50 g Na₂SO₄ getrocknet und am Wasserstrahlvakuum eingedampft. Das erhaltene Öl (95 g) wird unter leichtem Erwärmen in 250 ml Essigester gelöst und bei Raumtemperatur stehengelassen. Nach kurzer Zeit tritt Kristallisation ein. Man läßt etwa 4 Stunden bei Raumtemperatur und weitere 2 Stunden bei +4°C kristallisieren, dann saugt man den Feststoff ab und wäscht kurz mit ca. 100 ml Essigester.
Ausbeute: 31 g farblose Kristalle.
DC: Kieselgel, Essigester; Rf = 0,50.The evaporation residue obtained according to Example 1 (compound 2) is dissolved in 2 l of methanol, 2 l of 0.1 N H₂SO₄ and stirred for 1 hour under reflux. Then it is extracted once with 1.8 l and once with 600 ml of chloroform, the combined extracts are washed twice with 1 l of water, dried with 50 g of Na₂SO₄ and evaporated in a water jet vacuum. The oil obtained (95 g) is dissolved in 250 ml of ethyl acetate with gentle heating and left to stand at room temperature. Crystallization occurs after a short time. The mixture is left to crystallize for about 4 hours at room temperature and for a further 2 hours at + 4 ° C., then the solid is filtered off with suction and washed briefly with about 100 ml of ethyl acetate.
Yield: 31 g of colorless crystals.
TLC: silica gel, ethyl acetate; R f = 0.50.
28,1 g (65 mmol) der nach Beispiel 2 erhaltenen Verbindung (3) werden in 250 ml Tetrahydrofuran (THF) suspendiert und im Verlauf von 5 Stunden 69 ml (0,65 mol) Diazoessigester in 50 ml THF unter Rühren zugetropft. Zum Reaktionsstart und nach jeweils 1 Stunde werden je 50 mg Rhodium(II)-acetat zugegeben. Da dabei eine leichte Erwärmung der Reaktionslösung auftritt, empfiehlt es sich, die Lösung mit einem Wasserbad (25°C) zu kühlen. Man läßt 16 Stunden bei Raumtemperatur rühren, dann werden nach den oben beschriebenen Verfahren nochmals 69 ml Diazoessigester und insgesamt 250 g Rhodiumacetat zugegeben. Nach weiteren 16 Stunden wird der gesamte Vorgang ein drittes Mal wiederholt. Man läßt anschließend noch 1 Tag nachreagieren, versetzt dann mit 250 ml Methanol und dampft die Lösung im Vakuum ein. Der ölige Rückstand wird dreimal mit je 1 l Petrolether digeriert und nach Dekantieren in 100 ml Chloroform/Essigester = 2/1 gelöst. Man gibt das Rohprodukt auf eine Kieselgel-Säule (8,5 x 50 cm) und eluiert mit Chloroform/Essigester = 2/1. Die das reine Produkt (4) enthaltenden Fraktionen werden vereinigt, und das Lösungsmittel im Wasserstrahlvakuum entfernt.
Ausbeute: 18,2 g zähes Öl.
DC: Kieselgel, Essigester/Petrolether 2:1, Rf = 0,60.28.1 g (65 mmol) of the compound (3) obtained according to Example 2 are suspended in 250 ml of tetrahydrofuran (THF) and 69 ml (0.65 mol) of diazoacetic ester in 50 ml of THF are added dropwise over the course of 5 hours with stirring. At the start of the reaction and after 1 hour each, 50 mg of rhodium (II) acetate are added. As the reaction solution heats up slightly, it is advisable to cool the solution with a water bath (25 ° C). The mixture is stirred at room temperature for 16 hours, then 69 ml of diazoacetic ester and a total of 250 g of rhodium acetate are again added by the processes described above. After a further 16 hours, the entire process is repeated a third time. The mixture is then left to react for a further day, then 250 ml of methanol are added and the solution is evaporated in vacuo. The oily residue is digested three times with 1 liter of petroleum ether and, after decanting, dissolved in 100 ml of chloroform / ethyl acetate = 2/1. The crude product is placed on a silica gel column (8.5 x 50 cm) and eluted with chloroform / ethyl acetate = 2/1. The fractions containing the pure product (4) are combined and the solvent is removed in a water jet vacuum.
Yield: 18.2 g of viscous oil.
TLC: silica gel, ethyl acetate / petroleum ether 2: 1, R f = 0.60.
15,5 g (30 mmol) der nach Beispiel 3 erhaltenen Verbindung (4) löst man in 470 ml Methanol und versetzt mit einer Lösung von 6,05 g (60 mmol) KHCO₃ in 100 ml Wasser. Man läßt unter Rückfluß rühren und kontrolliert den Umsatz halbstündlich mittels DC. Wenn die Konzentration des Edukts nur noch ca. 5 % beträgt (etwa nach 3,5 Stunden), wird die Reaktion abgebrochen. Man stellt den pH mit Eisessig auf 5,0, dampft das Methanol im Wasserstrahlvakuum ab und verdünnt mit Wasser auf ca. 500 ml. Das Rohprodukt wird mit zweimal je 200 ml Essigester extrahiert, mit 200 ml Wasser gewaschen und die organische Lösung mit 30 g Na₂SO₄ getrocknet. Nach dem Eindampfen löst man die verbleibenden 11 g Öl in 40 ml Essigester/Eisessig = 9/1 und läßt bei Raumtemperatur stehen. Nach kurzer Zeit tritt Kristallisation ein. Man kühlt noch 1 Stunde bei +4°C, saugt den Feststoff ab und wäscht mit ca. 20 ml Essigester/Eisessig = 9/1 nach. Man erhält 3 g Produkt (5), das im Exsikkator über KOH getrocknet wird.15.5 g (30 mmol) of the compound (4) obtained according to Example 3 is dissolved in 470 ml of methanol and mixed a solution of 6.05 g (60 mmol) of KHCO₃ in 100 ml of water. The mixture is stirred under reflux and the conversion is checked every half hour by means of TLC. When the concentration of the starting material is only about 5% (after about 3.5 hours), the reaction is stopped. The pH is adjusted to 5.0 with glacial acetic acid, the methanol is evaporated off in a water jet vacuum and diluted with water to about 500 ml. The crude product is extracted twice with 200 ml of ethyl acetate, washed with 200 ml of water and the organic solution with 30 g Na₂SO₄ dried. After evaporation, the remaining 11 g of oil are dissolved in 40 ml of ethyl acetate / glacial acetic acid = 9/1 and left to stand at room temperature. Crystallization occurs after a short time. The mixture is cooled for 1 hour at + 4 ° C., the solid is filtered off with suction and washed with about 20 ml of ethyl acetate / glacial acetic acid = 9/1. 3 g of product (5) are obtained, which is dried over KOH in a desiccator.
Die Mutterlauge wird eingedampft und der Rückstand (ca. 7,5 g) auf eine Kieselgel-Säule (8,5 x 40 cm) aufgetragen. Nach Elution mit Essigester/Eisessig = 9/1 und Eindampfen der entsprechenden Fraktionen werden nochmals 2,2 g Produkt (5) erhalten, das noch Spuren von 12-O-Acetyl-digoxigenin-3-cme enthalten kann.
Ausbeute: 5,2 g farbloser Feststoff.
DC: Kieselgel, Essigester/Eisessig 9:1, Rf = 0,42.The mother liquor is evaporated and the residue (approx. 7.5 g) is applied to a silica gel column (8.5 x 40 cm). After elution with ethyl acetate / glacial acetic acid = 9/1 and evaporation of the corresponding fractions, another 2.2 g of product (5) are obtained, which may still contain traces of 12-O-acetyl-digoxigenin-3-cme.
Yield: 5.2 g of colorless solid.
TLC: silica gel, ethyl acetate / glacial acetic acid 9: 1, R f = 0.42.
4,49 g (10 mmol) der nach Beispiel 4 erhaltenen Digoxigenin-Carbonsäure (5) werden zusammen mit 1,27 g (11 mmol) N-Hydroxysuccinimid in 140 ml wasserfreiem THF gelöst und mit einer Lösung von 2,27 g (11 mmol) N,N′-Dicyclohexylcarbodiimid in 20 ml wasserfreiem THF versetzt. Man läßt 20 Stunden bei Raumtemperatur rühren, dann filtriert man vom ausgefallenen Harnstoff ab und dampft die Lösung im Wasserstrahlvakuum ein. Den Rückstand löst man in 150 ml Essigester, filtriert und wäscht mit 100 ml Wasser. Die organische Lösung wird danach sofort mit 5 g Na₂SO₄ getrocknet und eingedampft. Man löst das Rohprodukt in ca. 30 ml Essigester, filtriert und gießt unter Rühren langsam in 200 ml Diisopropylether. Der ausgefallene Ester (6) wird abgesaugt und im Exsikkator über Phosphorpentoxid getrocknet.
Ausbeute: 5,1 g farbloses Pulver.
DC: Kieselgel RP-18, Nitromethan/Ethanol 9:1; Rf = 0,66.4.49 g (10 mmol) of the digoxigenin carboxylic acid (5) obtained according to Example 4 together with 1.27 g (11 mmol) of N-hydroxysuccinimide in 140 ml of anhydrous THF dissolved and mixed with a solution of 2.27 g (11 mmol) of N, N'-dicyclohexylcarbodiimide in 20 ml of anhydrous THF. The mixture is stirred at room temperature for 20 hours, then the precipitated urea is filtered off and the solution is evaporated in a water jet vacuum. The residue is dissolved in 150 ml of ethyl acetate, filtered and washed with 100 ml of water. The organic solution is then immediately dried with 5 g Na₂SO₄ and evaporated. The crude product is dissolved in about 30 ml of ethyl acetate, filtered and slowly poured into 200 ml of diisopropyl ether with stirring. The precipitated ester (6) is filtered off and dried over phosphorus pentoxide in a desiccator.
Yield: 5.1 g of colorless powder.
TLC: silica gel RP-18, nitromethane / ethanol 9: 1; R f = 0.66.
272 mg (0,5 mmol) des nach Beispiel 5 erhaltenen Aktivesters (6) werden in 10 ml Dioxan gelöst und mit einer Lösung von 161 mg (0,55 mmol) N-(4-Azidobenzoyl)-1,8-diamino-3,6-dioxaoctan in 5 ml Wasser versetzt. Man läßt 2 Stunden bei Raumtemperatur rühren, dann dampft man das Dioxan im Wasserstrahlvakuum ab und verdünnt mit 50 ml Wasser. Die wäßrige Phase wird mit zweimal je 50 ml Essigester extrahiert, die vereinigten organischen Extrakte mit 5 g Na₂SO₄ getrocknet und eingedampft. Den Rückstand digeriert man mit 100 ml Diisopropylether, saugt ab und trocknet im Exsikkator über CaCl₂.
Ausbeute: 216 mg farbloses Pulver.
DC: Kieselgel RP-18, Nitromethan/Ethanol 9:1; Rf = 0,36.272 mg (0.5 mmol) of the active ester (6) obtained according to Example 5 are dissolved in 10 ml of dioxane and with a solution of 161 mg (0.55 mmol) of N- (4-azidobenzoyl) -1,8-diamino- 3,6-dioxaoctane in 5 ml of water. The mixture is stirred for 2 hours at room temperature, then the dioxane is evaporated off in a water jet vacuum and diluted with 50 ml of water. The aqueous phase is extracted twice with 50 ml of ethyl acetate, the combined organic extracts dried with 5 g Na₂SO₄ and evaporated. The residue is digested with 100 ml of diisopropyl ether, suction filtered and dried in a desiccator over CaCl₂.
Yield: 216 mg of colorless powder.
TLC: silica gel RP-18, nitromethane / ethanol 9: 1; R f = 0.36.
Das folgende Reaktionsschema B zeigt schematisch die schrittweise Herstellung von Dig-11-dUTP, das z.B. als Markerhapten zum Nachweis von Nukleinsäuren durch Hybridisierung mit einer komplementären, markierten Nukleinsäure verwendet werden kann, gemäß den Beispielen 7 bis 9.
C₃₁H₄₇NO₈ MG: 561.3C₃₁H₄₇NO₈ MG: 561.3
In einem 250 ml-Rundkolßen werden 465 mg Digoxigenin-3-carboxymethylether-N-hydroxysuccinimidester (8) (0,85 mmol) in 15 ml Dimethylformamid (DMF) gelöst und dazu eine Suspension von 112 mg 6-Aminocapronsäure (0,85 mmol) und 0,12 ml Triethylamin in 2 ml DMF gegeben. Man rührt über Nacht bei Raumtemperatur magnetisch, wobei allmählich eine homogene Lösung entsteht. Nach dieser Zeit ist die Umsetzung laut Dünnschichtchromatographie (Kieselgel; Essigsäureethylester/Petrolether/Ethanol 1:1:1, Detektion: Besprühen mit einer Mischung von 10 ml Eisessig + 0,2 ml konz. H₂SO₄ + 0,1 ml Anisaldehyd und Erhitzen auf 120°C bis zum Erscheinen blauschwarzer Flecke; Rf ca. 0,7; Rf Digoxigenin-OSu-ester ca. 0,85) praktisch vollständig.465 mg of digoxigenin-3-carboxymethyl ether-N-hydroxysuccinimide ester (8) (0.85 mmol) are dissolved in 15 ml of dimethylformamide (DMF) in a 250 ml round-bottomed flask, and a suspension of 112 mg of 6-aminocaproic acid (0.85 mmol ) and 0.12 ml of triethylamine in 2 ml of DMF. The mixture is stirred magnetically at room temperature overnight, gradually forming a homogeneous solution. After this time, the reaction is according to thin layer chromatography (silica gel; ethyl acetate / petroleum ether / ethanol 1: 1: 1, detection: spraying with a mixture of 10 ml glacial acetic acid + 0.2 ml conc. H₂SO₄ + 0.1 ml anisaldehyde and heating to 120 ° C until blue-black spots appear; R f approx. 0.7; R f digoxigenin-OSu-ester approx. 0.85) practically completely.
Man destilliert DMF im Hochvakuum restlos ab und löst das verbleibende Öl in 5 ml H₂O unter Zugabe von konz. Ammoniaklösung. Dann wird durch Zufügen von 22,5 ml wäßriger Zitronensäurelösung (100 g Zitronensäure/l) die "freie" Digoxigeninamidocapronsäure abgeschieden. Die harzig-zähe Masse wird durch Anreiben mit Wasser fest; man saugt ab, wäscht mehrfach mit H₂O nach und trocknet letztlich über P₂O₅ im Ölpumpenvakuum. Ausbeute: 325 mg = 68 % der Th.DMF is completely distilled off under high vacuum and the remaining oil is dissolved in 5 ml of H₂O with the addition of conc. Ammonia solution. The "free" digoxigenin amidocaproic acid is then separated off by adding 22.5 ml of aqueous citric acid solution (100 g of citric acid / l). The resinous and viscous mass is solidified by rubbing with water; it is suctioned off, washed several times with H₂O and finally dried over P₂O₅ in an oil pump vacuum. Yield: 325 mg = 68% of th.
C₃₅H₅₀N₂O₁₀ MG: 658.8C₃₅H₅₀N₂O₁₀ MG: 658.8
In einem 100 ml-Rundkolben werden 320 mg Digoxigenin-3-carboxymethylether- ε -amidocapronsäure (9) (0,57 mmol) in 2 ml wasserfreiem Dimethylformamid (DMF) gelöst, und nacheinander mit 70 mg N-Hydroxysuccinimid (0,6 mmol), sowie 130 mg Dicyclohexylcarbodiimid (0,63 mmol) versetzt. Man rührt über Nacht bei Raumtemperatur, saugt anderntags vom abgeschiedenen Dicyclohexylharnstoff ab und zieht das DMF im Ölpumpenvakuum ab. Das zurückbleibende Öl wird in 2 ml Essigsäureethylester aufgenommen und in ca. 15 ml eiskalten (-20°C) Petrolether eingerührt. Das ausgefallene, anfangs noch harzig-zähe Produkt reibt man mehrfach mit eiskaltem trockenem Petrolether bis zum Festwerden durch. Nach Trocknung über P₂O₅ im Vakuum erhält man 315 mg = 84 % der Th.
(Dig-11-dUTP)
C₄₃H₆₁N₄Na₄O₂₁P₃
MG: 1154,7 MG: 1154,7(Dig-11-dUTP)
C₄₃H₆₁N₄Na₄O₂₁P₃
MG: 1154.7 MG: 1154.7
245 mg Digoxigenin-3-carboxymethylether- ε -amidocapronsäure-N-hydroxysuccinimidester (10) (0,37 mmol) werden in 7 ml DMF gelöst und zu einer Lösung von 20 mg 5-Allylamino-2′-desoxy-uridin-5′-triphosphat-Tetralithiumsalz (0,37 mmol) in 6 ml H₂O gegeben. Man fügt dem Gemisch 6,2 ml 0,1 mol/l Natriumboratpuffer, pH 8,5 zu und rührt bei Raumtemperatur über Nacht (ca. 15 Stunden).245 mg of digoxigenin-3-carboxymethyl ether-ε-amidocaproic acid-N-hydroxysuccinimide ester (10) (0.37 mmol) are dissolved in 7 ml of DMF and to a solution of 20 mg of 5-allylamino-2'-deoxy-uridine-5 ' triphosphate tetralithium salt (0.37 mmol) in 6 ml of H₂O. 6.2 ml of 0.1 mol / l sodium borate buffer, pH 8.5 are added to the mixture and the mixture is stirred at room temperature overnight (about 15 hours).
In der Papierelektrophorese (0,05 mol/l Citratpuffer, pH 5,0) beobachtet man im UV-Licht nach dieser Zeit neben etwas unumgesetztem Allylamino-dUTP einen etwas tiefer laufenderen Fleck der gewünschten Verbindung (alternativ: Dünnschichtchromatographie (DC) auf Kieselgel, Fließmittel Isobuttersäure/konz. Ammoniaklösung/H₂O = 66:1:33, Detektion im UV oder Besprühen mit Anisaldehyd-Reagenz - siehe Beispiel 7 -; Rf-Werte: 5-Allylamino-dUTP 0,2; Dig-amidocapronsäure-OSu-ester 0,7; Dig-11-dUTP 0,45).In paper electrophoresis (0.05 mol / l citrate buffer, pH 5.0), in addition to a little unreacted allylamino-dUTP, a slightly deeper spot of the desired compound is observed in UV light after this time (alternatively: thin-layer chromatography (TLC) on silica gel, Plasticizer isobutyric acid / concentrated ammonia solution / H₂O = 66: 1: 33, detection in the UV or spraying with anisaldehyde reagent - see Example 7 -; R f values: 5-allylamino-dUTP 0.2; Dig-amidocaproic acid-OSu- ester 0.7; Dig-11-dUTP 0.45).
Zur Aufreinigung wird das Reaktionsgemisch im Ölpumpenvakuum bis zum festen Rückstand eingedampft, in 200 ml H₂O aufgenommen und auf eine Ionenaustauschersäule (DEAE-Sephadex A25, HCO
Analytik:H₂O-Bestimmung: 7,9 %
For purification, the reaction mixture is evaporated in an oil pump vacuum to a solid residue, taken up in 200 ml of H₂O and on an ion exchange column (DEAE-Sephadex A25, HCO
Analytics: H₂O determination: 7.9%
Dig-11-dUTP kann nach der "Random primed"-DNA-Markierungsmethode (DNA-Labeling and Detection Kit Nonradioactive, Best.Nr. 1093657, Fa. Boehringer Mannheim; Feinberg, A.P. und Vogelstein, B., Anal. Biochem. 132, (1983) 6) in eine Nukleinsäure eingeführt werden, wobei eine Nukleinsäuresonde, die Digoxigenin als Markierungshapten enthält, zum Nachweis dazu komplementärer Nukleinsäuren erhalten wird.Dig-11-dUTP can be carried out according to the "random primed" DNA labeling method (DNA labeling and detection kit non-radioactive, order no. 1093657, from Boehringer Mannheim; Feinberg, AP and Vogelstein, B., Anal. Biochem. 132 , (1983) 6) are introduced into a nucleic acid, a nucleic acid probe containing digoxigenin as labeling hapten being obtained for the detection of complementary nucleic acids.
Claims (11)
dadurch gekennzeichnet,
daß Z die Gruppe -COOH, -CN, -COOR⁵, -CONR¹R², -COONR³R⁴, -CON₃, -COOCOO-R⁵ oder -COOCO-R⁵, worin R¹,R²,R³ und R⁴ unabhängig voneinander ein Wasserstoffatom, eine Alkylgruppe, eine Aralkylgruppe oder eine Arylgruppe sind, oder R¹ und R² oder R³ und R⁴ zusammen auch ein carbo- oder heterocyclisches Ringsystem bilden können, und R⁵ eine Alkyl- oder Aralkylgruppe ist.2 digoxigenin derivatives of the formula (I) according to Claim 1,
characterized,
that Z is the group -COOH, -CN, -COOR⁵, -CONR¹R², -COONR³R⁴, -CON₃, -COOCOO-R⁵ or -COOCO-R⁵, wherein R¹, R², R³ and R⁴ independently of one another are a hydrogen atom, an alkyl group, an aralkyl group or are an aryl group, or R¹ and R² or R³ and R⁴ together can also form a carbo- or heterocyclic ring system, and R⁵ is an alkyl or aralkyl group.
dadurch gekennzeichnet,
daß Z bedeutet: -COOH, -COOC₂H₅
characterized,
that Z means: -COOH, -COOC₂H₅
dadurch gekennzeichnet,
daß n = 1 ist.4. digoxigenin derivatives of the formula (I) according to any one of claims 1 to 3,
characterized,
that n = 1.
dadurch gekennzeichnet,
daß man im 12-O-Acetyl-digoxigenin die freie OH-Gruppe in 3-Stellung durch Umsetzung mit Diazoessigester in an sich bekannter Weise in eine Verbindung der Formel (I) überführt, worin Z = COOC₂H₅ ist, und, wenn erwünscht, in dem erhaltenen Reaktionsprodukt in an sich bekannter Weise die COOC₂H₅-Gruppe in die COOH-Gruppe oder in eine andere funktionelle Carbonsäuregruppe Z überführt, und, wenn erwünscht, in den so erhaltenen Reaktionsprodukten die Gruppe Z in an sich bekannter Weise in eine andere Gruppe Z überführt.5. A process for the preparation of the digoxigenin derivatives of the formula (I) according to one of claims 1 to 4,
characterized,
that in 12-O-acetyl-digoxigenin the free OH group in the 3-position by reaction with diazoacetic ester is converted in a manner known per se into a compound of the formula (I), in which Z = COOC₂H,, and, if desired, in the reaction product obtained converts the COOC₂H₅ group into the COOH group or into another functional carboxylic acid group Z in a manner known per se, and, if desired, the group Z is converted into another group Z in a manner known per se in the reaction products thus obtained .
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| AT89119938T ATE102948T1 (en) | 1988-10-27 | 1989-10-26 | NEW DIGOXIGENIN DERIVATIVES AND THEIR USE. |
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE3836656 | 1988-10-27 | ||
| DE3836656A DE3836656A1 (en) | 1988-10-27 | 1988-10-27 | NEW DIGOXIGENINE DERIVATIVES AND THEIR USE |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| EP0371262A1 true EP0371262A1 (en) | 1990-06-06 |
| EP0371262B1 EP0371262B1 (en) | 1994-03-16 |
Family
ID=6366051
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| EP89119938A Expired - Lifetime EP0371262B1 (en) | 1988-10-27 | 1989-10-26 | Digoxigenin derivatives and their use |
Country Status (6)
| Country | Link |
|---|---|
| US (1) | US5198537A (en) |
| EP (1) | EP0371262B1 (en) |
| JP (1) | JPH0637513B2 (en) |
| AT (1) | ATE102948T1 (en) |
| DE (2) | DE3836656A1 (en) |
| ES (1) | ES2063098T3 (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6008334A (en) * | 1996-07-24 | 1999-12-28 | The Board Of Regents Of The University Of Oklahoma | Base-protected nucleotide analogs with protected thiol groups |
| EP0927180B1 (en) * | 1996-09-12 | 2007-08-08 | Roche Diagnostics GmbH | Heterocyclic compounds and their use for isolating nucleic acids |
| WO2018122043A1 (en) * | 2016-12-27 | 2018-07-05 | F. Hoffmann-La Roche Ag | Novel biotin-specific monoclonal antibody and use thereof |
| WO2018122204A1 (en) * | 2016-12-27 | 2018-07-05 | F. Hoffmann-La Roche Ag | Novel biotin-specific monoclonal antibody and use thereof |
| WO2018122205A3 (en) * | 2016-12-27 | 2018-08-09 | F. Hoffmann-La Roche Ag | Novel biotin-specific monoclonal antibody and use thereof |
Families Citing this family (53)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE3813278A1 (en) * | 1988-01-12 | 1989-07-20 | Boehringer Mannheim Gmbh | METHOD FOR DETECTING NUCLEIC ACIDS |
| DE4222254A1 (en) * | 1992-07-07 | 1994-01-13 | Bayer Ag | Photochemical labeling of nucleic acids with digoxigenin reagents and their use in gene probe test systems |
| US5518901A (en) * | 1993-04-19 | 1996-05-21 | Murtagh; James J. | Methods for adapting nucleic acid for detection, sequencing, and cloning using exonuclease |
| US6541608B1 (en) * | 1999-02-23 | 2003-04-01 | Baylor College Of Medicine | T cell receptor Vβ-Dβ-Jβ sequence and methods for its detection |
| EP1159457B1 (en) | 1999-02-23 | 2010-11-03 | Baylor College Of Medicine | T cell receptor vbeta - dbeta - jbeta) sequence and methods for its detection |
| US6232076B1 (en) | 2000-02-04 | 2001-05-15 | Genaissance Pharmaceuticals, Inc. | Stabilizer of dye sequencing products |
| JP2005172694A (en) * | 2003-12-12 | 2005-06-30 | Fuji Photo Film Co Ltd | Protein detection method |
| US20060069043A1 (en) * | 2004-09-29 | 2006-03-30 | Somberg John C | Chiral separation, characterization and biological action of optically active isomers of digoxin |
| SG162795A1 (en) | 2005-06-15 | 2010-07-29 | Callida Genomics Inc | Single molecule arrays for genetic and chemical analysis |
| WO2007044245A2 (en) | 2005-10-07 | 2007-04-19 | Callida Genomics, Inc. | Self-assembled single molecule arrays and uses thereof |
| CN101432439B (en) * | 2006-02-24 | 2013-07-24 | 考利达基因组股份有限公司 | High throughput genome sequencing on DNA arrays |
| SG10201405158QA (en) * | 2006-02-24 | 2014-10-30 | Callida Genomics Inc | High throughput genome sequencing on dna arrays |
| US7910354B2 (en) * | 2006-10-27 | 2011-03-22 | Complete Genomics, Inc. | Efficient arrays of amplified polynucleotides |
| EP2078197B1 (en) | 2006-11-01 | 2016-03-23 | Ventana Medical Systems, Inc. | Haptens, hapten conjugates, compositions thereof and method for their preparation and use |
| US20090111705A1 (en) * | 2006-11-09 | 2009-04-30 | Complete Genomics, Inc. | Selection of dna adaptor orientation by hybrid capture |
| US7682789B2 (en) * | 2007-05-04 | 2010-03-23 | Ventana Medical Systems, Inc. | Method for quantifying biomolecules conjugated to a nanoparticle |
| EP3561513A1 (en) * | 2007-05-23 | 2019-10-30 | Ventana Medical Systems, Inc. | Polymeric carriers for immunohistochemistry and in situ hybridization |
| ES2557594T3 (en) | 2008-06-05 | 2016-01-27 | Ventana Medical Systems, Inc. | Method for histochemical processing and the use of a composition for histochemical processing |
| US9050375B2 (en) | 2009-07-06 | 2015-06-09 | Hoffmann-La Roche, Inc. | Bi-specific digoxigenin binding antibodies |
| USPP22463P3 (en) * | 2010-02-16 | 2012-01-17 | Menachem Bornstein | Gypsophila plant named ‘Pearl Blossom’ |
| JP2013531801A (en) | 2010-07-02 | 2013-08-08 | ヴェンタナ メディカル システムズ， インク． | Hapten conjugates for target detection |
| US8865404B2 (en) | 2010-11-05 | 2014-10-21 | President And Fellows Of Harvard College | Methods for sequencing nucleic acid molecules |
| JP2014502607A (en) | 2011-01-03 | 2014-02-03 | エフ．ホフマン−ラ ロシュ アーゲー | Pharmaceutical composition of complex of digoxigenin conjugated with anti-DIG antibody and peptide |
| US10501791B2 (en) | 2011-10-14 | 2019-12-10 | President And Fellows Of Harvard College | Sequencing by structure assembly |
| WO2013184754A2 (en) | 2012-06-05 | 2013-12-12 | President And Fellows Of Harvard College | Spatial sequencing of nucleic acids using dna origami probes |
| EP2869848B1 (en) | 2012-07-04 | 2016-09-21 | F. Hoffmann-La Roche AG | Covalently linked antigen-antibody conjugates |
| WO2014006123A1 (en) | 2012-07-04 | 2014-01-09 | F. Hoffmann-La Roche Ag | Anti-biotin antibodies and methods of use |
| CN107082810B (en) | 2012-07-04 | 2020-12-25 | 弗·哈夫曼-拉罗切有限公司 | Anti-theophylline antibodies and methods of use |
| US9476089B2 (en) | 2012-10-18 | 2016-10-25 | President And Fellows Of Harvard College | Methods of making oligonucleotide probes |
| US9540685B2 (en) | 2013-03-15 | 2017-01-10 | President And Fellows Of Harvard College | Methods of identifying homologous genes using FISH |
| CN105873616B (en) | 2014-01-03 | 2020-06-05 | 豪夫迈·罗氏有限公司 | Covalently linked polypeptide toxin-antibody conjugates |
| RU2694981C2 (en) | 2014-01-03 | 2019-07-18 | Ф. Хоффманн-Ля Рош Аг | Covalently linked conjugates chelicar-antibody against chelicar and use thereof |
| JP6476194B2 (en) | 2014-01-03 | 2019-02-27 | エフ．ホフマン−ラ ロシュ アーゲーＦ． Ｈｏｆｆｍａｎｎ−Ｌａ Ｒｏｃｈｅ Ａｋｔｉｅｎｇｅｓｅｌｌｓｃｈａｆｔ | Bispecific anti-hapten / anti-blood brain barrier receptor antibodies, complexes thereof, and their use as blood brain barrier shuttles |
| CA2958292A1 (en) | 2014-08-19 | 2016-02-25 | President And Fellows Of Harvard College | Rna-guided systems for probing and mapping of nucleic acids |
| JP7038209B2 (en) | 2017-11-13 | 2022-03-17 | エフ．ホフマン－ラ ロシュ アーゲー | Equipment for sample analysis using epitaco electrophoresis |
| WO2020074742A1 (en) | 2018-10-12 | 2020-04-16 | F. Hoffmann-La Roche Ag | Detection methods for epitachophoresis workflow automation |
| US20220325268A1 (en) | 2019-05-14 | 2022-10-13 | Roche Sequencing Solutions, Inc | Devices and methods for sample analysis |
| CN112552367B (en) * | 2020-12-11 | 2023-06-06 | 郑州安图生物工程股份有限公司 | Digoxin derivative and preparation method thereof |
| EP4278181A1 (en) | 2021-01-15 | 2023-11-22 | Ventana Medical Systems, Inc. | Storage stable caged haptens |
| US20220228200A1 (en) | 2021-01-19 | 2022-07-21 | 10X Genomics, Inc. | Methods and compositions for internally controlled in situ assays |
| WO2022256324A1 (en) | 2021-06-01 | 2022-12-08 | 10X Genomics, Inc. | Methods and compositions for analyte detection and probe resolution |
| CN117751197A (en) | 2021-06-02 | 2024-03-22 | 10X基因组学有限公司 | Sample analysis using asymmetric circularizable probes |
| US20230026886A1 (en) | 2021-07-13 | 2023-01-26 | 10X Genomics, Inc. | Methods for preparing polymerized matrix with controllable thickness |
| WO2023015192A1 (en) | 2021-08-03 | 2023-02-09 | 10X Genomics, Inc. | Nucleic acid concatemers and methods for stabilizing and/or compacting the same |
| US20230061542A1 (en) | 2021-08-16 | 2023-03-02 | 10X Genomics, Inc. | Probes comprising a split barcode region and methods of use |
| US20230242974A1 (en) | 2021-12-27 | 2023-08-03 | 10X Genomics, Inc. | Methods and compositions for rolling circle amplification |
| US20230279475A1 (en) | 2022-01-21 | 2023-09-07 | 10X Genomics, Inc. | Multiple readout signals for analyzing a sample |
| WO2023164570A1 (en) | 2022-02-23 | 2023-08-31 | Insitro, Inc. | Pooled optical screening and transcriptional measurements of cells comprising barcoded genetic perturbations |
| WO2023215603A1 (en) | 2022-05-06 | 2023-11-09 | 10X Genomics, Inc. | Methods and compositions for in situ analysis of v(d)j sequences |
| WO2023215612A1 (en) | 2022-05-06 | 2023-11-09 | 10X Genomics, Inc. | Analysis of antigen and antigen receptor interactions |
| US20240035071A1 (en) | 2022-06-17 | 2024-02-01 | 10X Genomics, Inc. | Catalytic de-crosslinking of samples for in situ analysis |
| US20240101978A1 (en) | 2022-08-12 | 2024-03-28 | 10X Genomics, Inc. | Puma1 polymerases and uses thereof |
| WO2024040060A1 (en) | 2022-08-16 | 2024-02-22 | 10X Genomics, Inc. | Ap50 polymerases and uses thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB2003480A (en) * | 1977-09-01 | 1979-03-14 | Squibb & Sons Inc | Digoxigenin derivatives |
| EP0173251A2 (en) * | 1984-08-28 | 1986-03-05 | Roche Diagnostics GmbH | Nucleic-acids sequence derivatives, process for their preparation and their use in the determination of nucleic acids |
| EP0174753A1 (en) * | 1984-08-23 | 1986-03-19 | Becton Dickinson and Company | Amplified assay for a ligand |
Family Cites Families (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US3855208A (en) * | 1971-05-24 | 1974-12-17 | Becton Dickinson Co | Derivatives of digoxigenin |
| US3925355A (en) * | 1974-03-01 | 1975-12-09 | Corning Glass Works | Labelled digoxin derivatives for radioimmunoassay |
| US4064227A (en) * | 1975-03-17 | 1977-12-20 | Mallinckrodt, Inc. | Radioimmunoassay method for the determination of cardiotonic glycosides |
| US4082747A (en) * | 1975-10-01 | 1978-04-04 | Mallinckrodt, Inc. | Chemical process |
| JPS53119873A (en) * | 1977-03-28 | 1978-10-19 | Rohm & Haas | Digoxigenin derivative |
| US4469797A (en) * | 1982-09-23 | 1984-09-04 | Miles Laboratories, Inc. | Digoxigenin immunogens, antibodies, labeled conjugates, and related derivatives |
-
1988
- 1988-10-27 DE DE3836656A patent/DE3836656A1/en not_active Withdrawn
-
1989
- 1989-10-25 US US07/427,249 patent/US5198537A/en not_active Expired - Lifetime
- 1989-10-26 EP EP89119938A patent/EP0371262B1/en not_active Expired - Lifetime
- 1989-10-26 AT AT89119938T patent/ATE102948T1/en not_active IP Right Cessation
- 1989-10-26 ES ES89119938T patent/ES2063098T3/en not_active Expired - Lifetime
- 1989-10-26 DE DE89119938T patent/DE58907232D1/en not_active Expired - Lifetime
- 1989-10-27 JP JP1278799A patent/JPH0637513B2/en not_active Expired - Lifetime
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB2003480A (en) * | 1977-09-01 | 1979-03-14 | Squibb & Sons Inc | Digoxigenin derivatives |
| EP0174753A1 (en) * | 1984-08-23 | 1986-03-19 | Becton Dickinson and Company | Amplified assay for a ligand |
| EP0173251A2 (en) * | 1984-08-28 | 1986-03-05 | Roche Diagnostics GmbH | Nucleic-acids sequence derivatives, process for their preparation and their use in the determination of nucleic acids |
Non-Patent Citations (1)
| Title |
|---|
| EXPERIENTIA, Band 36, Oktober 1980, Seiten 1141-1143, Basel, CH; N. MONJI et al.: "Quantification of digoxin by enzyme immunoassay: synthesis of a maleimide derivative of digoxigenin succinate for enzyme coupling" * |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6008334A (en) * | 1996-07-24 | 1999-12-28 | The Board Of Regents Of The University Of Oklahoma | Base-protected nucleotide analogs with protected thiol groups |
| US6107039A (en) * | 1996-07-24 | 2000-08-22 | The Board Of Regents Of The University Of Oklahoma | Assays using base protected table 1 |
| EP0927180B1 (en) * | 1996-09-12 | 2007-08-08 | Roche Diagnostics GmbH | Heterocyclic compounds and their use for isolating nucleic acids |
| WO2018122043A1 (en) * | 2016-12-27 | 2018-07-05 | F. Hoffmann-La Roche Ag | Novel biotin-specific monoclonal antibody and use thereof |
| WO2018122204A1 (en) * | 2016-12-27 | 2018-07-05 | F. Hoffmann-La Roche Ag | Novel biotin-specific monoclonal antibody and use thereof |
| WO2018122205A3 (en) * | 2016-12-27 | 2018-08-09 | F. Hoffmann-La Roche Ag | Novel biotin-specific monoclonal antibody and use thereof |
| CN110088139A (en) * | 2016-12-27 | 2019-08-02 | 豪夫迈·罗氏有限公司 | New biotin monoclonal antibody specific and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| DE58907232D1 (en) | 1994-04-21 |
| EP0371262B1 (en) | 1994-03-16 |
| DE3836656A1 (en) | 1990-05-03 |
| ES2063098T3 (en) | 1995-01-01 |
| US5198537A (en) | 1993-03-30 |
| JPH02191298A (en) | 1990-07-27 |
| ATE102948T1 (en) | 1994-04-15 |
| JPH0637513B2 (en) | 1994-05-18 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| EP0371262B1 (en) | Digoxigenin derivatives and their use | |
| EP0618231B1 (en) | Immunologically active conjugates and a method for their preparation | |
| DE3856358T3 (en) | Chemiluminescent esters, thioesters and amides that can be used for tests | |
| EP1386158B1 (en) | Object comprising an uncharged, functionalized hydrogel surface | |
| DE3033691A1 (en) | DIAMETRIC ACIDS TIED TO A SUBSTRATE AND THEIR CHELATES | |
| DE3628573C2 (en) | Chemiluminescent acridine derivatives, processes for their preparation and their use in luminescence immunoassays | |
| DE2910998A1 (en) | PROCESSES FOR THE MANUFACTURE OF BOUND ENZYME CONTAINING PRODUCTS AND THEIR USE | |
| DE2928048C2 (en) | ||
| EP0632810B1 (en) | New biotinylation reagent | |
| EP0451810B1 (en) | Hapten-biotin-conjugates and their use | |
| DE3919915A1 (en) | AMINOALKYLMALEIMIDES AND DERIVED HAPTEN AND ANTIGEN DERIVATIVES AND CONJUGATES WITH PEPTIDES OR PROTEINS | |
| DE2921782C2 (en) | ||
| EP0747447A2 (en) | Novel oxazine dyes and their use as fluorescent label | |
| DE3129705C2 (en) | ||
| EP0378203B1 (en) | Cobalamine-acid hydrazides and cobalamine conjugates derived therefrom | |
| EP0747699A1 (en) | Reagent for suppressing interference for use in the measurement of an analyte using a luminescent metal complex | |
| DE2600835A1 (en) | DIGOXIN DERIVATIVES, THE PROCESS FOR THEIR MANUFACTURING AND THEIR USE FOR RADIO-IMMUNESTIMATION OF DIGOXIN | |
| EP1308728A2 (en) | Method and compounds for the fluorescent labelling of biomolecules and polymer particles | |
| DE2924332C2 (en) | ||
| DE2537129C3 (en) | Reactive asymmetrical dicarboxylic acid esters containing a digoxin or digitoxin residue, processes for their preparation and their use for the preparation of test reagents | |
| DE2354253C2 (en) | Process for the preparation of a reagent for the radioimmunological determination of testosterone | |
| DE2616724C3 (en) | Hydroxylalkylamides of hydroxyphenylalanines, a process for their preparation and their use | |
| AT362391B (en) | METHOD FOR PRODUCING NEW CONNECTIONS FOR DETECTING HEART GLYCOSIDES | |
| DE4343261C2 (en) | Process for the preparation of raw materials for immunoassays and bioassays | |
| CH587865A5 (en) | Reagent for radio-immunoassay of testosterone - by reacting testerone-oxime with tyrosine ester |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PUAI | Public reference made under article 153(3) epc to a published international application that has entered the european phase |
Free format text: ORIGINAL CODE: 0009012 |
|
| 17P | Request for examination filed |
Effective date: 19891026 |
|
| AK | Designated contracting states |
Kind code of ref document: A1 Designated state(s): AT BE CH DE ES FR GB GR IT LI LU NL SE |
|
| 17Q | First examination report despatched |
Effective date: 19930120 |
|
| GRAA | (expected) grant |
Free format text: ORIGINAL CODE: 0009210 |
|
| AK | Designated contracting states |
Kind code of ref document: B1 Designated state(s): AT BE CH DE ES FR GB GR IT LI LU NL SE |
|
| REF | Corresponds to: |
Ref document number: 102948 Country of ref document: AT Date of ref document: 19940415 Kind code of ref document: T |
|
| GBT | Gb: translation of ep patent filed (gb section 77(6)(a)/1977) |
Effective date: 19940315 |
|
| REF | Corresponds to: |
Ref document number: 58907232 Country of ref document: DE Date of ref document: 19940421 |
|
| ITF | It: translation for a ep patent filed |
Owner name: ST. CONSUL.BREVETTUALE S.R.L. |
|
| REG | Reference to a national code |
Ref country code: GR Ref legal event code: FG4A Free format text: 3011464 |
|
| ET | Fr: translation filed | ||
| REG | Reference to a national code |
Ref country code: ES Ref legal event code: FG2A Ref document number: 2063098 Country of ref document: ES Kind code of ref document: T3 |
|
| PLBE | No opposition filed within time limit |
Free format text: ORIGINAL CODE: 0009261 |
|
| STAA | Information on the status of an ep patent application or granted ep patent |
Free format text: STATUS: NO OPPOSITION FILED WITHIN TIME LIMIT |
|
| EAL | Se: european patent in force in sweden |
Ref document number: 89119938.2 |
|
| 26N | No opposition filed | ||
| REG | Reference to a national code |
Ref country code: GB Ref legal event code: IF02 |
|
| REG | Reference to a national code |
Ref country code: CH Ref legal event code: PFA Owner name: BOEHRINGER MANNHEIM GMBH Free format text: BOEHRINGER MANNHEIM GMBH#SANDHOFERSTRASSE 112-132 POSTFACH 31 01 20#D-68298 MANNHEIM (DE) -TRANSFER TO- BOEHRINGER MANNHEIM GMBH#SANDHOFERSTRASSE 112-132 POSTFACH 31 01 20#D-68298 MANNHEIM (DE) |
|
| PGFP | Annual fee paid to national office [announced via postgrant information from national office to epo] |
Ref country code: NL Payment date: 20080915 Year of fee payment: 20 |
|
| PGFP | Annual fee paid to national office [announced via postgrant information from national office to epo] |
Ref country code: GB Payment date: 20080915 Year of fee payment: 20 |
|
| PGFP | Annual fee paid to national office [announced via postgrant information from national office to epo] |
Ref country code: DE Payment date: 20081031 Year of fee payment: 20 Ref country code: CH Payment date: 20081028 Year of fee payment: 20 Ref country code: LU Payment date: 20081022 Year of fee payment: 20 |
|
| PGFP | Annual fee paid to national office [announced via postgrant information from national office to epo] |
Ref country code: AT Payment date: 20080915 Year of fee payment: 20 Ref country code: ES Payment date: 20081013 Year of fee payment: 20 |
|
| PGFP | Annual fee paid to national office [announced via postgrant information from national office to epo] |
Ref country code: BE Payment date: 20081031 Year of fee payment: 20 Ref country code: SE Payment date: 20081007 Year of fee payment: 20 Ref country code: IT Payment date: 20081017 Year of fee payment: 20 |
|
| PGFP | Annual fee paid to national office [announced via postgrant information from national office to epo] |
Ref country code: FR Payment date: 20081006 Year of fee payment: 20 |
|
| PGFP | Annual fee paid to national office [announced via postgrant information from national office to epo] |
Ref country code: GR Payment date: 20081014 Year of fee payment: 20 |
|
| REG | Reference to a national code |
Ref country code: CH Ref legal event code: PL |
|
| BE20 | Be: patent expired |
Owner name: *BOEHRINGER MANNHEIM G.M.B.H. Effective date: 20091026 |
|
| REG | Reference to a national code |
Ref country code: GB Ref legal event code: PE20 Expiry date: 20091025 |
|
| EUG | Se: european patent has lapsed | ||
| REG | Reference to a national code |
Ref country code: ES Ref legal event code: FD2A Effective date: 20091027 |
|
| NLV7 | Nl: ceased due to reaching the maximum lifetime of a patent |
Effective date: 20091026 |
|
| PG25 | Lapsed in a contracting state [announced via postgrant information from national office to epo] |
Ref country code: ES Free format text: LAPSE BECAUSE OF EXPIRATION OF PROTECTION Effective date: 20091027 |
|
| PG25 | Lapsed in a contracting state [announced via postgrant information from national office to epo] |
Ref country code: NL Free format text: LAPSE BECAUSE OF EXPIRATION OF PROTECTION Effective date: 20091026 |
|
| PG25 | Lapsed in a contracting state [announced via postgrant information from national office to epo] |
Ref country code: GB Free format text: LAPSE BECAUSE OF EXPIRATION OF PROTECTION Effective date: 20091025 |